PEPT1, a proton-coupled oligopeptide transporter (POT) of the SLC15 family, has nutritional importance because of its intestinal absorption of small peptides from the diet and because of its reabsorption of peptide-bound amino nitrogen from glomerular filtrate in kidney. PEPT1 also has pharmacological significance in its ability to transport therapeutic agents (e.g., ?-lactam antibiotics, angiotensin-converting enzyme inhibitors, antiviral nucleoside prodrugs) and potentially toxic peptidomimetics (e.g., 5-aminolevulinic acid). However, other members of the POT family (i.e., PEPT2, PHT1 and PHT2) are expressed in the intestine and kidney, thereby confounding an accurate assessment of its role and relevance. Our laboratory has recently established a colony of PEPT1 knockout mice, a unique resource in which to validate in situ mechanistic studies with in vivo whole animal experiments. The availability of these knockout mice is particularly exciting given the recent association of aberrant PEPT1 colonic expression and inflammatory bowel disease progression. With this in mind, the long-term objectives of this resubmission application are to define the physiological, pharmacological, and pathological roles and relevance of PEPT1. Our working hypothesis is that PEPT1 is a critical transporter in the intestinal absorption, disposition, and dynamics of peptides and peptide-like drugs, and that it plays an important role in the pathogenesis of intestinal inflammation. To test this hypothesis, the following specific aims are proposed: Aim 1. To determine the in situ intestinal transport properties of peptide-like drugs (and prodrugs), bacterially-derived peptides, and anti- inflammatory tripeptides; Aim 2. To characterize the in vivo absorption and disposition of peptide-like drugs (and prodrugs), and anti-inflammatory tripeptides; and Aim 3. To evaluate the role and relevance of PEPT1 in mediating the inflammatory response to bacterially-derived peptides and anti-inflammatory tripeptides. By combining cellular/tissue, molecular, and whole animal studies in wild-type and PEPT1 null mice, the proposed studies will greatly advance our understanding of the in vivo role, significance and vectorial transport of peptides, peptide-like drugs (and prodrugs) by PEPT1 (as opposed to other transporters and/or passive processes). Moreover, our findings will provide important new insights into the mechanisms of PEPT1- mediated intestinal inflammation and, as a result, identify a new target for therapeutic strategies against inflammatory bowel disease. Finally, the proposed studies may offer rare insight into the variability of peptide/mimetic kinetics and response in those human subjects with genetic polymorphisms.